The long-term goal of this project is to understand how oligodendrocytes, the myelinating glial cells of the central nervous system (CNS), are produced. During development, dividing neuroepithelial precursors give rise to oligodendrocyte progenitor cells (OPCs). Following migration to become dispersed throughout the CNS, some OPCs differentiate as myelinating oligodendrocytes but others remain in a non-myelinating state into adulthood. The mechanisms that guide formation of OPCs from proliferating neuroepithelial precursors and specify myelinating and non-myelinating fates are not well known. Using zebrafish as a model system, this project combines in vivo live cell imaging with genetic and pharmacological manipulations to test novel hypotheses formulated to identify mechanisms that regulate OPC specification. Specific Aim 1 will test the hypothesis that microRNA inhibition of apical Par polarity proteins regulates the proliferation to differentiation transition by making precursors less sensitive to Shh signaling. Specific Aim 2 will test the hypothesis that different levels of Shh signaling specify myelinating and non-myelinating OPCs. Specific Aim 3 will identify gene functions that mediate specification of myelinating and non-myelinating OPC fates in response to differential Shh signaling. Completion of these Aims will fill important gaps in our basic knowledge of neural development and reveal molecular targets that could be used to promote myelination following injury or disease.